Pyrimine-producing bacteria

ABSTRACT

A novel pyrimine-producing strain belonging to genus Pseudomonas exhibits the following bacteriological properties: denitrification reaction: negative; assimilation of carbon sources: 
     D-arabinose: positive 
     L-lysine: negative 
     and a novel pyrimine-producing strain belonging to genus Pseudomonas exhibits the following bacteriological properties: dentrification reaction: negative; assimilation of carbon sources: 
     D-arabinose: positive 
     L-lysine: positive 
     These novel strains produce pyrimine in high yield and if the strains are cultured in a proper culture medium in the presence of an iron salt, a natural red dye, ferropyrimine, can be easily produced and directly be recovered from the culture medium.

BACKGROUND OF THE INVENTION

The present invention relates to bacteria which can produce pyrimineuseful as an intermediate of a natural dye, ferropyrimine, and to amethod for effectively preparing pyrimine in which thepyrimine-producing bacteria are used.

Natural dyes have recently attracted much attention since doubt hasarisen regarding the safety of synthetic dyes. Among such natural dyes,a natural red dye, ferropyrimine, has been produced by culturing aGH-strain belonging to genus Pseudomonas in a culture medium to have thestrain produce pyrimine and simultaneously associate the resultingpyrimine with iron ions which are added to the culture medium in advanceand isolating the thus formed ferropyrimine from the culture medium (seeBiochemistry, 1965, 4, No. 10).

SUMMARY OF THE INVENTION

An object of the present invention is to provide novelpyrimine-producing bacteria.

Another object of the present invention is to provide a method foreffectively preparing pyrimine.

These and other objects of the present invention will be apparent fromthe following description and Examples.

According to one aspect of the present invention there is provided afirst and novel pyrimine-producing strain belonging to genus Pseudomonasexhibiting the following bacteriological properties:

Denitrification reaction: negative

Assimilation of carbon sources:

D-arabinose: positive

L-lysine: negative

According to another aspect of the present invention, there is provideda second and novel pyrimine-producing strain belonging to genusPseudomonas exhibiting the following bacteriological properties:

Dentrification reaction: negative

Assimilation of carbon sources:

D-arabinose: positive

L-lysine: positive

These strains produce pyrimine in an amount of not less than 400 μg/mlwhen they are cultured in the presence of a plant in an LS basal mediumand they start production of pyrimine at latest 8 days after theinitiation of the cultivation.

According to a further aspect of the present invention, there isprovided a method for preparing pyrimine which comprises the steps ofsubjecting a pyrimine-producing strain to a mixed cultivation in aculture medium in the presence of a plant and isolating pyrimineproduced from the culture medium.

According to another aspect of the present invention, there is provideda method for preparing pyrimine which comprises the steps of culturingpyrimine-producing bacteria belonging to genus Pseudomonas, in thepresence of a plant, in a culture medium containing sugar, a nitrogenatom-containing compound, a magnesium atom-containing compound, aphosphorus atom-containing compound and iron atom-containing compound,but free of calcium and chloride ions and then isolating the resultingpyrimine.

According to another aspect of the present invention, there is provideda method for preparing pyrimine which comprises the steps of culturingpyrimine-producing bacteria belonging to genus Pseudomonas in a culturemedium containing an organic nitrogen atom-containing compound, sugar,an inorganic nitrogen atom-containing compound, a phosphorusatom-containing compound and iron atom-containing compound, but free ofcalcium and chloride ions, and having a pH ranging from 3.8 to 5, andthen isolating the resulting pyrimine.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The first and novel pyrimine-producing strain of the present inventionhas the following bacteriological properties:

Bacteriological Properties 1. Morphological Properties

Morphology: bacillus which exists independently or is attached in theform of a chain and does not form a spore;

Mobility: It has motility and a polar flagellum;

Size: 0.6˜0.8μ×1.0˜3.5μ;

Gram-staining: negative;

2. Cultivability in Various Culture Mediums

(1) Bouillon Culture

Growth: moderate; Tunic: It forms a ring; Precipitates: slightlypresent; Turbidity: observed.

(2) Bouillon Slant Culture

Shape: fibrous; Surface: smooth and glossy; Periphery: It forms entirecolonies; Color Tone: milk-white; Transparency: opaque.

(3) Bouillon Agar Plate Culture

Shape: perfect circle; Periphery: It forms entire colonies; Shape ofSurface Protuberance: lens-like shape; Surface: smooth and glossy; ColorTone: milk-white;

(4) Bouillon Agar Stab Culture

It grows on the surface of the culture medium and along the upperportion of the stab.

3. Physiological Properties

(1) Behavior Against Oxygen: aerobic;

(2) Catalase: +

(3) Oxidase: +

(4) Arginine Dehydrolase: -

(5) Lipase: + (hydrolyzation of Tween 80)

(6) Lecithinase: - (decomposition of yolk)

(7) Hydrolyzation of Gelatin: -

(8) Hydrolyzation of Starch: -

(9) Hydrolyzation of Extracellular Poly-β-hydroxybutyric acid(PHB): -

(10) Autotrophic Growth with use of H₂ : -

(11) Formation of Dye: It does not form any dye in Pseudomonas F agarand Pseudomonas P agar.

(12) Reduction of Nitrates:

(13) Denitrification Reaction:

(14) Decomposition point in protocatechulate: ortho-position

(15) Intracellular Accumulation of PHB: +

(16) Formation of Levan from Sucrose: -

(17) Growth Temperature Range: from 11° to 40° C.; optimum growthtemperature: 30° to 32° C.

(18) Growth pH Range: from 3.8 to 8.5; optimum growth pH: 5.5 to 6.5.

(19) Assimilation of Carbon Sources:

    ______________________________________                                        D-glucose:   +       D-fructose:     +                                        D-arabinose: +       trehalose:      ±                                     mannitol:    +       inositol:       +                                        glycerol:    +       L-lysine:       -                                        L-valine:    ±    β-alanine: +                                        glycolate:   +       p-hydroxybenzoate:                                                                            +                                        ______________________________________                                    

The classification of the strain of the present invention was carriedout according to the method disclosed in "Bergey's Manual of SystematicBacteriology", 1984, Vol. 1 on the basis of the foregoingbacteriological properties. As a result, this strain is a gram-negativebacillus which shows motility by a polar flagellum, is aerobic, ispositive to catalase test and is positive to oxidase test. Therefore,this strain was judged to be a bacillus belonging to genus Pseudomonas.

Among strains belonging to genus Pseudomonas, Pseudomonas cepacia andPseudomonas gladioli are known as strains which intracellularlyaccumulate poly-β-hydroxybutyric acid as a carbon source-storagesubstance, grow at 40° C. and decomposes protocatechuic acid at theortho-position.

However, this strain cannot use sucrose, but can use glycine as a carbonsource, as will be shown below. The strain (Pseudomonas cepacia) differsfrom the strain of this invention (Pseudomonas sp. K-2) in these points.

    ______________________________________                                                         Pseudomonas Pseudomonas                                      Properties       sp. K-2     cepacia                                          ______________________________________                                        Assimilation of Hydrocarbon                                                                    -           +                                                Sucrose                                                                       Glycine          +           -                                                ______________________________________                                    

On the other hand, this strain cannot form levan from sucrose, cannothydrolyze gelatin and has no lecithinase as will be shown below. Thestrain (Pseudomonas gladioli) differs from the strain of this invention(Pseudomonas sp. K-2) in these points.

    ______________________________________                                                         Pseudomonas                                                                              Pseudomonas                                       Properties       sp. K-2    gladioli                                          ______________________________________                                        Formation of levan                                                                             -          +                                                 from sucrose                                                                  Hydrolyzation of gelatin                                                                       -          +                                                 Lecithimase      -          +                                                 ______________________________________                                    

Therefore, it is reasonable to judge that the present strain is a novelstrain belonging to genus Pseudomonas and was named Pseudomonas sp. K-2.The present strain is deposited with the Fermentation Research Institute(FRI) under the accession No. FERM BP-2933.

The second pyrimine-producing strain according to the present inventionhas the same bacteriological properties as those of the firstpyrimine-producing strain of the present invention except for thefollowing points:

Growth Temperature Range: 11° to 35° C.

Assimilation of Carbon Sources: L-lysine +

Therefore, this stain was judged to be a bacillus belonging to genusPseudomonas.

Among strains belonging to genus Pseudomonas, Pseudomonas solanacearumis known as strains which intracellularly accumulatespoly-β-hydroxybutyric acid as a carbon source-storage substance, doesnot grow at 40° C. and decomposes protocatechuic acid at theortho-position.

However, this strain (Pseudomonas solanacearum) can under undergo adenitrification reaction and cannot use D-arabinose as a carbon source,as will be shown below. The strain (Pseudomonas solanacearum) differsfrom the strain of this invention (Pseudomonas sp. K-1) in these points.

    ______________________________________                                                         Pseudomonas Pseudomonas                                      Properties       sp. K-1     solanacearum                                     ______________________________________                                        Denitrification Reaction                                                                       -           +                                                Assimilation of Hydrocarbon                                                                    +           -                                                D-arabinose                                                                   ______________________________________                                    

Therefore, it is reasonable to judge that the present strain is a novelstrain belonging to genus Pseudomonas and was named Pseudomonas sp. K-1.The present strain is deposited with the Fermentation Research Institute(FRI) under the accession No. FREM BP-2932.

When the foregoing strains, Pseudomonas sp. K-2 and Pseudomonas sp. K-1,are cultured, it is preferred that they are first subjected to apre-cultivation at a temperature of about 25° C. for 2 to 3 days in aculture medium such as a nutrient agar culture medium or an improved GSculture medium and then directly inoculated into an LS culture medium,but it is also possible to directly inoculate and culture the strains inan LS culture medium.

If the foregoing strains sp. K-2 and sp. K-1 or other pyrimine-producingstrains are cultured in the presence of a plant, pyrimine can beobtained in high efficiency. As such plants, there may be suitably used,for instance, plants of the Liliaceae family such as Welsh onion; plantsof the Cruciferae family such as lettuce, cabbage, Japanese dalehorseradish, Japanese radish and water cress; plants of the Perillafamily such as perilla, basil and mint; plants of the Polygonaceaefamily such as Rumex acetosa; plants of the Leguminosae family such asharicot bean; plants of the Solanaceae family such as tomato, eggplantand guinea pepper; and plants of the Gesneriaceae family such ascentoporia.

Sections of mesophyll, stem, rhizome or the like of these plants areinoculated into an LS agar culture medium to which a hormone such asα-naphthylacetic acid or kinetin is supplemented under sterilizedconditions and then the differentiation thereof is induced and theresulting product is preferably employed in the present invention assuch a plant. Alternatively, it is also preferable to use those obtainedby sowing seeds of such a plant which have been subjected tosterilization treatment in the same LS agar culture medium to thus havethem germinate and grow.

Moreover, it is further possible to use plant cells (such as cells oftobacco) and callus obtained by sterilizing a plant body, andinoculating the resulting pieces of explant into a culture medium understerile conditions to induce callus formation. The amount of the plantsto be added to the culture medium is not critical, but it in generalranges from 0.01 to 5 g and preferably 0.1 to 0.5 g per 100 parts byweight of the culture medium. In addition, the density of the strain tobe inoculated into the culture medium desirably ranges of not less than10⁵ /ml, more desirably about 10⁵ to 10⁷ /ml.

The culture medium used for subjecting the foregoing strains and theplants to a mixed cultivation comprises the following components whichare commonly incorporated into culture mediums:

(i) Carbon Sources: sugars such as glucose, fructose, sucrose and/orsugar alcohols such as glycerol;

(ii) Nitrogen Sources: inorganic nitrogen atom-containing compounds suchas ammonium salts and/or nitrates;

(iii) Inorganic Salts: phosphates, sulfates or the like of metals suchas potassium and magnesium.

In the present invention, it is advantageous that ferric ions are addedto the culture medium in advance in order to directly obtainferropyrimine from pyrimine produced by the strain during thecultivation thereof. Such iron ions can be supplied to the culturemedium in the form of salts such as ferric sulfate. The amount of theferric ions in the culture medium is desirably not less than 10 ppm andpreferably 20 to 100 ppm expressed in the amount of salts.

The culture mediums used for producing pyrimine may be either solid orliquid. Moreover, the strains of the present invention can be culturedby a variety of methods, but preferably they are cultured by a rotaryshaking culture or an aeration-agitation culture. In addition, thestrains are preferably cultured under aerobic conditions. For instance,they can be cultured while being irradiated with light rays. The cultureconditions are properly selected and adjusted so that the maximum amountof the intended pyrimine can be accumulated in the culture medium.Preferred culture conditions are as follows:

(i) pH of Initial Culture Medium: 3.8 to 7.0; more preferably 5.0 to6.0.

(ii) Cultivation Temperature Range: 15° to 30° C.; more preferably 20°to 25° C.

(iii) Cultivation Time: 8 to 20 days.

(iv) pH of Culture Medium during the culturing: 3.8 to 6.0, preferably4.0 to 4.5.

Any known means can be adopted for isolating and purifying the pyrimineproduced by the strain and accumulated in the culture medium accordingto the foregoing method.

The foregoing pyrimine-producing strains of the present invention andother pyrimine-producing strains are subjected to a mixed culture in thepresence of a plant in one of culture mediums explained above. However,the optimum yield of pyrimine can be achieved by use of a culture mediumwhich comprises a sugar, a nitrogen atom-containing compound, amagnesium atom-containing compound, a phosphorus atom-containingcompound and an iron atom-containing compound, but is free of calciumand chloride ions. More specifically, the culture medium used in thepresent invention comprises, for instance, 20 to 80 g, preferably 30 to60 g of sugar; 1.0 to 5.0 g, preferably 1.5 to 3.5 g of a nitrogenatom-containing compound; 0.1 to 0.5 g, preferably 0.3 to 0.4 g of amagnesium atom-containing compound; 0.1 to 0.8 g, preferably 0.15 to 0.5g of a phosphorus atom-containing compound; 0.01 to 0.2 g, preferably0.02 to 0.15 g of an iron atom-containing compound; and the balance ofwater. The culture medium may further comprises other components free ofcalcium and/or chloride ion-containing compounds. For instance, it maycomprise 0 to 0.2 g of an alkali metal salt of EDTA, 0 to 20 g of agaror the like.

Alternatively, pyrimine can also be obtained, in high yield, byculturing the pyrimine-producing strains, in the presence or absence ofa plant, in a culture medium which comprises at least one organicnitrogen atom-containing compound, for example, selected from the groupconsisting of meat extract, proteose peptone, casamino acids, powderysoy protein products and corn steep liquor; a sugar, an inorganicnitrogen atom-containing compound, a phosphorus atom-containing compoundand an iron atom-containing compound, but is free of calcium andchloride ions. Examples of the inorganic nitrogen atom-containingcompounds are ammonium salts and nitrates. The phosphorusatom-containing compound supplies phosphate ions to the culture mediumand typical examples thereof are potassium phosphate and sodiumphosphate. The iron atom-containing compound supplies iron ions to theculture medium and typical examples thereof include ferric sulfate. Ashas been explained above, if ferric ions are present in the culturemedium in advance, ferropyrimine can directly be recovered from theculture medium.

A specific example of such a culture medium comprises, per literthereof, 20 to 80 g, preferably 25 to 75 g of a sugar; 1 to 5 g,preferably 1.5 to 3.5 g of an inorganic nitrogen atom-containingcompound; 0.2 to 1 g, preferably 0.25 to 0.6 g of a phosphorusatom-containing compound; 0.01 to 0.2 g, preferably 0.02 to 0.15 g of aniron atom-containing compound; 0.1 to 4 g, preferably 0.2 to 2 g of anorganic nitrogen atom-containing compound; and the balance of water. Theculture medium of this kind may further comprise other components whichare free of calcium and chloride ions. For instance, it may comprise 0to 0.45 g of a magnesium atom-containing compound such as magnesiumsulfate; 0 to 0.2 g of an alkali metal salt of EDTA; 0 to 20 g of agarand so forth. In this connection, 1M potassium phosphate buffer (pH 5)is employed as the phosphorus atom-containing compound and, in thiscase, the buffer is used in an amount ranging from 100 to 200 g.

According to the present invention, there are provided novel strainsbelonging to genus Pseudomonas which can produce pyrimine in high yieldas well as a novel method for preparing pyrimine with the use of suchstrains. Moreover, if the strains are cultured in a proper culturemedium in the presence of iron salts, a natural red dye, i.e.,ferropyrimine, can be easily and directly obtained from the culturemedium.

The present invention will hereinafter be described in more detail withreference to the following non-limitative working Examples.

EXAMPLE 1

0.1 mg/l each of α-naphthylacetic acid and kinetin was added to aLinsmaier-Skoog basal medium (1650 mg of NH₄ NO₃ ; 1900 mg of KNO₃ ; 440mg of CaCl₂.2H₂ O; 370 mg of MgSO₄.7H₂ O; 170 mg of KH₂ PO₄ ; 6.2 mg ofH₃ BO₃ ; 22.3 mg of MnSO₄.4H₂ O; 8.6 mg of ZnSO₄.7H₂ O; 0.83 mg of KI;0.25 mg of Na₂ MoO₄.2H₂ O; 0.025 mg of CoCl₂.6H₂ O; 0.025 mg ofCuSO₄.5H₂ O; 37.3 mg of Na₂ -EDTA; 27.8 mg of FeSO₄.7H₂ O; 100 mg ofmyoinositol; 0.4 mg of thiamin hydrochloride; 30,000 mg of sucrose;1,000 ml of distilled water) and then the pH of the resulting solutionwas adjusted to 5.8 to obtain a culture medium (hereunder referred to as"LS culture medium"). The LS culture medium was dispensed as 50 mlportions into ten 300 ml volume Erlenmeyer flasks and the flasks weresealed with adsorbent wadding plugs to perform sterilization at 121° C.for 15 minutes. After cooling, there was inoculated, into the sterilizedLS culture medium, seedlings of horseradish which had been separatelygrown in an LS agar culture medium (0.8% of agar) under sterileconditions and a loopful of the strain, Pseudomonas sp. K-2, which hadbeen separately grown in an improved GS culture medium (the preparationthereof will be detailed below). The culture was performed at 25° C. inaccordance with a rotary shaking culture (150 rpm) under irradiationwith light rays.

After 17 days, the culture medium was centrifuged to remove the cellbodies or the like and the resulting supernatant was filtered through amembrane filter having a pore size of 0.45 μm. After the addition of anFeSO₄.7H₂ O solution (278 mg/l) to the filtrate, the absorbance thereofwas determined at 558 nm to estimate the amount of pyrimine present inthe culture medium in terms of a calibration curve and it was found tobe 1650 μg/ml. The production of pyrimine was started on the sixth dayof cultivation.

Improved GS Culture Medium

(i) A solution (a) was prepared so that it had the followingcomposition: 30,000 mg of glucose; 770 mg of meat extract (availablefrom Difco Laboratories); 1650 mg of NH₄ NO₃ ; 370 mg of MgSO₄.7H₂ O;27.8 mg of FeSO₄.7H₂ O; 37.3 mg of Na₂ -EDTA; 20,000 mg of agar; and1,000 ml of distilled water. The pH value thereof was controlled to 6.0by the addition of 1N KOH.

(ii) A solution (b): 1M potassium phosphate buffer (pH 6.0).

The improved GS culture medium was prepared by treating the solutions(a) and (b) in an autoclave at 121° C. for 15 minutes to performsterilization and mixing the autoclaved solutions (a) and (b) in a ratioof 2 ml of the solution (b) per 11 ml of the solution (a).

EXAMPLE 2

The same procedures used in Example 1 were repeated except that thestrain, Pseudomonas sp. K-1 was substituted for the strain Pseudomonassp. K-2 used in Example 1. After 17 days, the content of ferropyriminein the resulting culture medium was determined in terms of a calibrationcurve and it was found to be 1750 μg/ml. The production of pyrimine wasstarted on the fifth day of cultivation.

EXAMPLE 3

An improved S culture medium which comprised 30,000 mg of sucrose; 1650mg of NH₄ NO₃ ; 370 mg of MgSO₄.7H₂ O; 500 mg of KH₂ PO₄ ; 27.8 mg ofFeSO₄.7H₂ O; 37.3 mg of Na₂ -EDTA; and 1,000 ml of distilled water andhad a pH of 7.0 was dispensed as 50 ml portions into two 300 ml volumeErlenmeyer flasks and the flasks were sealed with adsorbent waddingplugs to perform sterilization at 121° C. for 15 minutes. After cooling,there were inoculated, into the sterilized LS culture medium, seedlingsof horseradish which had been separately grown in an LS agar culturemedium (0.8% of agar) under sterile conditions and a loopful of thestrain, Pseudomonas sp. K-1, which had been separately grown in animproved GS culture medium (the preparation thereof will be detailedbelow). The culture was performed at 25° C. in accordance with a rotaryshaking culture (150 rpm) while irradiating with light rays.

After 12 days, the culture medium was centrifuged to remove the cellbodies or the like, and the resulting supernatant was filtered through amembrane filter having a pore size of 0.45 μm. After the addition of anFeSO₄.7H₂ O solution (278 mg/l) to the filtrate, the absorbance thereofwas determined at 558 nm to evaluate the amount of ferropyrimine presentin the culture medium in terms of a calibration curve and it was foundto be 1360 μg/ml.

EXAMPLE 4

The same procedures used in Example 3 were repeated except that thestrain, Pseudomonas sp. K-2 was substituted for the strain Pseudomonassp. K-1 used in Example 3 and that after 13 days, the culture medium wascentrifuged to remove the cell bodies and the like. The content offerropyrimine in the resulting culture medium was found to be 940 μg/ml.

EXAMPLE 5

The following solutions were prepared:

(a) a solution of 36,000 mg of glucose in 300 ml of distilled water;

(b) a solution obtained by dissolving 1980 mg of NH₄ NO₃, 222 mg ofMgSO₄.7H₂ O, 300 mg of KH₂ PO₄, 16.68 mg of FeSO₄.7H₂ O and 22.38 mg ofNa₂ -EDTA in 300 ml of distilled water and then adjusting the pH thereofto 6.0 with 1N KOH;

(c) a solution of 1,200 mg of meat extract (available from DifcoLaboratories) in 120 ml of distilled water; and

(d) 120 ml of 1M potassium phosphate buffer (pH 5.0).

The foregoing solutions (a) to (d) were sterilized at 121° C. for 15minutes and introduced into a 2l volume jar fermenter (sterilized) tomix them under sterile conditions. Separately, the strain, Pseudomonassp. K-2 was pre-cultured at 25° C. for 48 hours by a shaking culturetechnique in a 300 ml volume Erlenmeyer flask containing a culturemedium which was prepared in the same manner and had the samecomposition as above. 84 ml of the precultured strain was inoculatedinto the culture medium contained in the jar fermenter and cultured at25° C. by an aeration-agitation culture (flow rate of air=1.5 l/min;rate of revolution=300 to 400 rpm).

After the second day of cultivation, the culture was performed whileadjusting the pH value of the culture medium to 4.2 to 4.5 by theaddition of 1N KOH and 1N phosphoric acid.

After 12 days, the culture medium was centrifuged to remove the cellbodies and the like, and the resulting supernatant was filtered througha membrane filter having a pore size of 0.45 μm. After the addition ofan FeSO₄.7H₂ O solution (278 mg/l) to the filtrate, the absorbancethereof was determined at 558 nm to evaluate the amount of ferropyriminepresent in the culture medium in terms of a calibration curve and it wasfound to be 1940 μg/ml.

EXAMPLE 6

The same procedures used in Example 5 were repeated except that thestrain, Pseudomonas sp. K-1 was substituted for the strain Pseudomonassp. K-2 used in Example 5 and that after 8 days, the culture medium wascentrifuged to remove the cell bodies or the like. The content offerropyrimine in the resulting culture medium was found to be 1950μg/ml.

What is claimed is:
 1. A bacteriologically pure culture of apyrimine-producing strain belonging to genus Pseudomonas exhibiting thefollowing bacteriological properties:Denitrification reaction: negativeAssimilation of carbon sources:D-arabinose: positive L-lysine: positiveWherein it produces pyrimine in an amount of not less than 400 μg/mlwhen it is cultured in the presence of a plant in an LS basal medium andit starts production of pyrimine at latest 8 days after the initiationof the culture.
 2. The strain of claim 1 wherein it is a strain FERMBP-2932.